This document discusses autoantibodies and methods for detecting anti-nuclear antibodies (ANA). ANAs are antibodies directed against nuclear and cytoplasmic antigens and are associated with various autoimmune diseases. The three main methods for detecting ANAs are indirect immunofluorescence assay using HEp-2 cells, ELISA, and multiplex bead immunoassays. The immunofluorescence assay is commonly used for initial screening due to its ability to detect multiple antigen patterns but has limitations. ELISA and bead assays allow detection of specific autoantibodies and have improved sensitivity and specificity compared to immunofluorescence.

1. ANA IN
AUTOIMMUNITY
- Dr. Anamika.
D

2. Autoantibodies
O Directed against
O Components of the cell surface,
O Cytoplasm components
O Nuclear components

3.  Antibodies to cell nucleus component
ANA, anti-dsDNA, antibodies to extractable
nuclear antigen (ENA), (anti-Sm, anti-RNP)
 Antibodies to cytoplasmic antigens
anti-SSA, ANCA
 Cell-specific autoantibodies
lymphocytotoxic antibodies, anti-neurone
antibodies, anti-erythrocyte antibodies, anti-
platelet antibodies
 Antibodies to serum components
antiphospholipid antibody,antiglobulin,
rheumatoid factor

4. Anti-nuclear Antibodies
O Also known as antinuclear factor or ANF
O 4 types:
O Anti- nuclear antibodies to DNA
O ANA to histones
O ANA to non-histone proteins bound to
RNA
O ANA to nucleolar antigens

5. Many sub-types
 anti-Ro/SS-A antibodies Sjogrens syndrome
 anti-La/SS-B antibodies
 anti-dsDNA antibodies
 anti-Sm antibodies SLE
 anti-nRNP antibodies – Mixed connective
tissue disease
 anti-Scl-70 antibodies - scleroderma
 Anti-Jo-1 antibodies – Polymyositis and
dermatomyositis
 anti-histone antibodies – drug induced lupus
 anti-centromere antibodies – CREST syndrome
 anti-sp100 antibodies – primary biliary
cirrhosis

6. ANA TESTING
Three main methods:
1. Indirect Immunofluorescence Assay
(IFA)
2. Enzyme-linked immunosorbent assay
(ELISA)
3. Multiplex Bead Immunoassays
O Microarrays

7. Sample preparation
O Collect blood specimens, usually from arm
of pt , at any time.
O No special instructions given
O Separate the serum.
O Specimens may be refrigerated at 2–8°C
for up to seven days or frozen for up to six
months. Avoid repetitive freezing and
thawing.

8. Indirect immunofluorescence
O Immunofluorescence is commonly used
O In the past patients serum was placed on
to slides with rodent (or other animal) cells
and IF was performed to look for
antibodies binding to cellular components
O What problems does this cause?

9. O Human and rodent cells differ (slightly),
and so some people with obvious
rheumatic disease would be negative on
this test. “ANA-negative lupus”
O Now there are human tumor cell lines that
are used (HEp-2 are preferred)

10. How is the test done?
O Whole HEp- 2 cells containing these antigens are attached to
microscope slide (cells fixed into separate dots on the slide)
O Patient serum is diluted and dropped onto HEp-2 slides
O If antibody is resent , it binds to antigen on Hep -2 cells
O Incubate for 20 mins at room temerature.
O Wash to remove unreacted antibody.
O Add secondary antibody -anti-human globulin labeled with
fluorescent tag or enzyme bind to pts antibody
characteristic fluorescent pattern
O Read using an IF scope

14. O Another source of false negatives includes
how the tissues are fixed onto the slides
O Ethanol and methanol fixation may
remove Ro/SSA antigens from cells, so
the cells are fixed with acetone

15. Results
 Depend on
 Titer of antibody (highest dilution of serum at which
auto- antibodies are still deectable)
 IFpattern
 Titers less than 1:40 should be considered
negative (20-30% of healthy people)
 Titers of 1:40 to 1:160 positive at low titer
(further workup is not recommended in the
absence of specific symptoms)

16. Results
O Titers equal to or greater than 1:160
positive

17. IF Patterns
O Peripheral or rim staining pattern = dsDNA
O Homogenous/ diffuse pattern = chromatin,dsDNA,
histones
O Speckled = many antigens like Sm antigen,
Ribonucleo-protein, SS-A& SS-B reactive antigens
O Nucleolar = RNA
O Centromeric = centromere

18. Ro
La
Smith
RNP
Jo-1
Scl-70
Ro
Nucleolar
dsDNA
Rim
Speckled
Homogenous
Nucleosomes
Ro
La
Smith
RNP
Jo-1
Scl-70
Ro
dsDNA
Nucleolar proteins

19. Homogenous pattern
 Smooth, even staining
of the nucleus with or
without
apparent masking of
the nucleoli
 Seen in Systemic lupus
– anti – DNA, anti-
histone

20. Rim/ Peripheral pattern
 Fluorescence is most
intense at the periphery of
the nucleus with a large
ring starting from the
internal nuclear membrane
and the rest of the nucleus
showing weaker yet
smooth staining.
 Not seen on Hep-2
Seen in SLE – anti
-DNA

21. Nucleolar pattern
O 23 or 46 (or some
multiple of 46) bright
speckles or ovoid
granules spread over
the nucleus of
interphase cells
O Seen in Diffuse
Scleroderma
O Scl-70

23. Speckled pattern
O Large speckles
covering the whole
nucleoplasm,
interconnected by a
fine fluorescent
network.
O m/c observed
O Least specific
O Seen in sjogrens
syndrome, SLE,
polymyositis,
dermtomyositis

25. Patterns

26. IFA- False Negatives:
o Rodent / animal cells used
o Methanol /ethanol fixation
o In Sjogren’s Syndrome,polymyositis, and
dermatomyositis (ANA -ve in >50%)
o If single antibody, at very low level, is
present: - SSA, subacute cutaneous lupus
- dsDNA, ANA negative lupus

27. False Positives:
O 33% of normals can be positive
O > 20% healthy relatives
O 75% elderly population
O In other diseases: viral infections, cirhosis,
Chronic Pulmonary Fibrosis, Chronic Infection,
Chronic Hepatitis ,Cancer

28. ELISA
O Also k/a ANA BLOT test or ANA ELISA TEST
O Amount of antibodies in units per given amount of
blood
O Whole Hep-2 cells are lysed, centrifuged to
concentrate the nuclei
O Other purified antigens are added, boost signal
for all autoantigens (e.g., SSA, Scl-70, Jo-1)
O Coated onto high-affinity binding wells to retain
all signals during processing

29. O Diluted human serum is added to wells coated
with purified nuclear antigens.
O ANA IgG specific antibody, if present, binds to
the antigen.
O All unbound materials are washed away and the
enzyme conjugate is added to bind to the
antibody-antigen complex, if present.
O Excess enzyme conjugate is washed off and
substrate is added.
O The plate is incubated to allow the hydrolysis of
the substrate by the enzyme.
O The intensity of the color generated is
proportional to the amount of IgG specific
antibody in the sample.

30. O ELISA detects the same ANA antibodies
as IFA, with improved sensitivity/specificity
O Results in one determination (no
repeating to titrate)
O Objective, automatable, requires no
specialized training
O ELISA reports out Index Values

31. Multiplex Bead Immunoassay
O Individual antigens, cell components are
coated onto multiple beads
O All activities are detected and identified in
parallel
O High cost of test and automation

32. To summarize…
O You screen for ANAs using IF on slides
with HEp-2 cells
O If it’s positive look for the specific
antigen using ELISA
O We don’t screen for ANAs using ELISA
because it’s hard to get all the various
antigens (40+) onto the well walls